Bacteria exhibit a rich diversity of morphologies. Within this diversity, there is a uniformity of shape for each species that is replicated faithfull each generation, suggesting that bacterial shape is as selectable as any other biochemical adaptation. In fact, for several bacterial species, specific morphologies dictate adhesion properties and pathogenicity. Therefore, determining how bacteria actively restructure their morphologies in response to environmental conditions, or how they have evolved various morphologies over time, can help direct efforts towards impeding their persistence, proliferation, and pathogenicity. The goal of this application is to exploit a nonessential form of zonal growth, the stalk, in the genera of Caulobacterales, in order to dissect the process of stalk positioning and synthesis and to identify genes involved in stalk synthesis. The stalk provides an excellent system for studying the general question of how bacterial cells drive peptidoglycan synthesis in specific zones to regulate growth and to generate precise morphologies. Phylogenic studies have indicated that within Caulobacterales, stalk positioning and number have diversified in the Asticcacaulis genus from the ancestral, single polar stalk to a single sub-polar stalk in A. excentricus and subsequently to two bilateral stalks in A. biprosthecum. Moreover, the natural variation and evolution of stalk positioning in these species correlates with the localization of SpmX, a developmental regulator that has been coopted for stalk synthesis in A. excentricus and A. biprosthecum. This application proposes to (1) identify and map the regions of SpmX that are critical for subcellular localization and recruitment of the stalk synthesis machinery in Asticcacaulis, (2) determine the role of the SpmX muramidase domain in localization and stalk synthesis, and (3) identify binding partners of SpmX in the Asticcacaulis genus that either target SpmX to its sub-polar or bilateral positions or perform as stalk initiation and elongation factors. These aims will combine reverse and forward genetic approaches with fluorescent microscopy and electron cryotomography to dissect the structure and function of SpmX in order to determine its role as a stalk positioning and synthesis factor as well as to investigate how it has evolved new functions in the Asticcacaulis genus. In addition, SpmX will be used to identify other stalk positioning and synthesis factors. Overall, the results of this proposal will (1) develp our working knowledge of stalk synthesis, (2) inform how bacteria remodel the cell wall at the molecular level, and (3) help to resolve how prokaryotes evolve novel morphologies. Because peptidoglycan is unique to bacteria and critical for their survival, the most widely used and effective antibiotics are those that target peptidoglycan synthesis. Therefore strengthening our understanding of peptidoglycan synthesis and how it is utilized to maintain bacterial cell shape is a major task in developing new antibiotics and strategies for controlling bacterial populations.